1. Field of the Invention
The present invention relates generally to conjugate molecules and more specifically, to conjugate molecules and their use in the delivery of active agents into cells using endogenous cellular transport pathways.
2. Background Information
Transporter proteins are involved in the cellular uptake of various molecules into and/or through cells. Carrier-mediated transport systems use proteins that are anchored to the cell membrane, typically by a plurality of membrane-spanning domains and function by transporting their substrates via active or passive mechanisms. Carrier-mediated transport systems are involved in the active or non-active, facilitated transport of many important nutrients such as vitamins, sugars, and amino acids. Carrier-mediated transporters are also present in organs such as the liver and kidney, in which the proteins are involved in the excretion or re-absorption of circulating compounds. Polar or hydrophilic compounds typically diffuse poorly across the lipid bilayers that constitute cellular membranes. For many small molecules (e.g., amino acids, di- and tripeptides, monosaccharides, nucleosides and water-soluble vitamins) there exist specific carrier-mediated transporters for active transport of the solute molecules across biological membranes.
The uptake or release physiological nucleosides and many of their synthetic analogs by mammalian cells occurs primarily by means of specific carrier-mediated transporters known as nucleoside transporters. Nucleoside transporters have been classified into two categories: (i) equilibrative (facilitated diffusion) and (ii) concentrative (secondary active) sodium-dependent. Two equilibrative transport systems with similar broad substrate specificities have been identified and designated as the es (equilibrative sensitive) and ei (equilibrative insensitive) transporters, on the basis of their sensitivity or insensitivity to inhibition by nitrobenzylthioinosine (NBMPR, 1), respectively. As many as six sodium ion-coupled (concentrative) nucleoside transport systems designated cif/N1, cit/N2, cib/N3, cit/N4, cs/N5 and csg/N6 have also been functionally identified in mammalian tissues.
The anti-DNA antibody fragment 3E10 Fv has been demonstrated to be a novel molecular delivery vehicle due to its penetration into living cells with specific nuclear localization, absence of toxicity, and successful delivery of therapeutic cargo proteins in vitro and in vivo. Elucidation of the pathway that allows 3E10 Fv to cross cell membranes is critical to the development of new molecular therapies, which rely on the regulation of gene expression by intranuclear transduction of macromolecules.